2011 Annual Report
1a.Objectives (from AD-416)
The long-term objective of this project is to characterize and develop rapid molecular-based protocols for detecting high-risk foreign plant pathogenic bacteria. During the next 5 years we will focus on collecting germplasm, characterize, fingerprint, and determine the taxonomy of high-risk foreign and domestic bacteria which may threaten U.S. agriculture through natural or deliberate introduction. We will collect cultures of Burkolderia andropogonis, B. glumae, B. caryophylli and B. gladioli and conduct sequencing, fingerprinting, and determine taxonomy. Citrus seedlings (infected with the non-cultureable Candidatus Liberibacter asiaticus, L. africanus, and L. americanus) will be collected, and we will develop a means to grow these fastidious organisms. Same-day on-site molecular techniques will be developed for rapid identification and detection of B. andropogonis, glumae, B. caryophylli, B. gladioli, L. asiaticus, L. africanus, and L. americanus.
1b.Approach (from AD-416)
Collect and add new germplasm of domestic and foreign sources to the International Collection of Phytopathogenic Bacteria maintained at ARS/Ft. Detrick. Develop improved biosensors, DNA-based techniques including real-time PCR, and other novel techniques for rapid, sensitive detection and identification of bacteria. Determine the genetic profiles of foreign bacteria using molecular techniques, including DNA/DNA hybridization, AFLP, pulse gel electrophoresis, and sequencing. Various media designed for growing insect bacteria and phytoplasma will be tested for culturing Liberibacter.
To improve growth of Liberibacter on artificial media, we continued to test compounds identified from citrus leaf petiole extracts and phloem cells. New cultures have been initiated with FL strains of L. asiaticus for inoculation of citrus at Ft. Pierce, FL and subsequent demonstration of Koch's postulates in the fall of 2011. Polyclonal antibodies generated against cultivated Liberibacter cells were developed and tested in Western blot and ELISA formats. The antibodies demonstrated specificity for a protein antigen protein found in leaves of infected citrus plants, but not in control (uninfected) plants. The antibodies should be applicable for use in immunoflourescence assays for diagnosing HLB in citrus and psyllid vectors.